馮 路, 薛躍華, 徐正保, 嚴秀友, 方立仁
(臺州市中心醫(yī)院神經(jīng)外科, 浙江 臺州 318000)
NF-κB抑制制對腦出血大鼠神經(jīng)損傷的影響
馮 路△, 薛躍華, 徐正保, 嚴秀友, 方立仁
(臺州市中心醫(yī)院神經(jīng)外科, 浙江 臺州 318000)
目的探討核因子κB(NF-κB)抑制劑吡咯烷二硫代氨基甲酸鹽(PDTC)對腦出血(ICH)大鼠神經(jīng)功能及神經(jīng)細胞凋亡的調(diào)節(jié)作用。方法取SPF級SD大鼠隨機分假手術(shù)組(sham組)、ICH組、PDTC低濃度組(Plow組)和PDTC高濃度組(Phigh組),每組6只。采用自體血注入法建立大鼠ICH模型,Plow組和Phigh組在缺血后2 h分別給予100 mg/kg和200 mg/kg的PDTC腹腔注射,sham組和ICH組給予等體積的生理鹽水;24 h后,采用改良的Longa分級法進行神經(jīng)功能評分;TUNEL法測定神經(jīng)細胞凋亡情況;并檢測腦組織中丙二醛(MDA)的含量和超氧化物歧化酶(SOD)的活性。此外,采用Western blot法檢測腦組織中p-P65及cleaved caspase-3的蛋白水平。結(jié)果與sham組相比,ICH組大鼠的神經(jīng)功能評分顯著升高(P<0.05);應(yīng)用PDTC后,Plow組和Phigh組動物的神經(jīng)功能評分都有所降低,但2組間差異無統(tǒng)計學意義。與sham組相比,ICH組大鼠神經(jīng)細胞凋亡明顯增多(P<0.05);應(yīng)用PDTC后,2組動物神經(jīng)細胞凋亡數(shù)目顯著降低,且Phigh組的細胞凋亡數(shù)目顯著低于Plow組(P<0.05)。與sham組相比,ICH組大鼠腦組織的MDA含量升高,SOD活性降低(P<0.05);應(yīng)用PDTC后,2組動物腦組織中的MDA含量顯著降低,而SOD活性升高,且這一趨勢在Phigh組表現(xiàn)更為明顯。與sham組相比,ICH組大鼠腦組織中p-P65和cleaved caspase-3的蛋白水平顯著增加(P<0.05);應(yīng)用PDTC后,2組動物腦組織中p-P65和cleaved caspase-3的蛋白水平顯著降低,且Phigh組中p-P65和cleaved caspase-3的蛋白水平顯著低于Plow組。結(jié)論NF-κB抑制劑PDTC能減輕腦出血后繼發(fā)性腦損傷,且大劑量作用效果較好;其作用機制可能與降低MDA含量、提高SOD活性進而抑制神經(jīng)細胞凋亡有關(guān)。
核因子κB抑制劑; 細胞凋亡; 腦出血; 氧化應(yīng)激
腦出血(intracerebral hemorrhage,ICH)是常見的腦血管疾病,其致殘率和死亡率一直高居不下,成為危害人類健康的重要因素[1]。研究顯示,腦出血導致的腦損傷主要體現(xiàn)在2個方面:一是原發(fā)性損傷,即血腫的占位效應(yīng)和血腫對周圍腦組織產(chǎn)生的直接破壞作用;二是腦出血后的繼發(fā)性損傷,如腦水腫、細胞凋亡及壞死和腦組織嚴重的炎癥反應(yīng)等[2-3]。而繼發(fā)性損傷是導致病程遷延,影響患者預后的重要原因。研究顯示,腦出血后產(chǎn)生大量的自由基,并激活細胞凋亡信號,對腦組織造成不可逆的損傷[3-4],故減少神經(jīng)細胞的凋亡對減輕繼發(fā)性腦損傷、恢復腦組織功能具有重要的臨床意義。
核因子κB(nuclear factor κB,NF-κB)屬于轉(zhuǎn)錄因子蛋白家族,廣泛存在于體內(nèi)多種細胞中。靜息狀態(tài)下,NF-κB與其抑制蛋白(inhibitor of NF-κB, IκB)形成復合體,不具備活性;一旦接受刺激,IκB磷酸化后暴露NF-κB的核定位位點,NF-κB轉(zhuǎn)移入核,并誘導相關(guān)基因的表達[5-6]。目前,NF-κB在中樞神經(jīng)系統(tǒng)中的研究主要集中于炎癥反應(yīng)和免疫反應(yīng)。Zhang等[7]的研究顯示,腦出血患者血腫周圍組織中NF-κB的活性與患者預后密切相關(guān)。已有研究證實ICH所致的繼發(fā)性腦損傷中確實包括了炎癥反應(yīng)的相關(guān)機制,但是NF-κB在ICH所致神經(jīng)細胞凋亡中的作用尚缺乏直接的證據(jù)。本研究以NF-κB為切入點,探討NF-κB抑制劑吡咯烷二硫代氨基甲酸鹽(pyrrolidine dithiocarbamate, PDTC)對腦出血大鼠神經(jīng)功能及神經(jīng)細胞凋亡的調(diào)節(jié)作用及相關(guān)機制。
健康雄性SD大鼠27只,體重260~280 g,購自臺州醫(yī)學院實驗動物中心,許可證號:SYXK(浙)2014-0006。飼養(yǎng)、手術(shù)、給藥及取材等環(huán)節(jié)均嚴格遵守實驗動物科學與管理的有關(guān)規(guī)定。
腦立體定向儀購自廣東省深圳瑞沃德生命科技有限公司。NF-κB抑制劑PDTC購自Sigma;TUNEL法凋亡細胞原位末端標記試劑盒購于南京凱基生物科技發(fā)展有限公司;抗p-P65、P65、cleaved caspase-3、caspase-3和GAPDH抗體購于Santa Cruz;丙二醛(malondialdehyde,MDA)及超氧化物歧化酶(superoxide dismutase,SOD)檢測試劑盒購于南京建成生物工程研究所。
2.1模型的制備和分組 將實驗動物隨機分為2組:假手術(shù)(sham)組6只和手術(shù)組21只。ICH模型的制備采用自體血注入法:術(shù)前12 h開始禁食,麻醉后將大鼠仰臥位固定,保證其前后囟處于同一水平;備皮并沿頭頂部正中矢狀位切開皮膚,鈍性分離前囟,于前囟前0.2 mm,中線右旁3 mm處定位鉆孔,直徑0.5 mm,進針約5.5 mm深(即尾殼核)。同時將大鼠尾部置于40 ℃溫水中5 min,消毒后迅速剪去5 mm鼠尾,用微量注射器取血50 μL,并勻速(10 μL/min)向腦內(nèi)注入自體血,注血后留針5 min,緩慢退針,局部骨蠟封閉后逐層縫合皮膚即可。假手術(shù)組僅在尾殼核部位鉆孔不注血。模型參照Bederson等[8]的評定方法檢測動物行為,出現(xiàn)手術(shù)對側(cè)前肢凹收、屈曲于腹下,同側(cè)肢體伸展等體征則視為造模成功。最終造模成功19只,依據(jù)體重納入18只,并隨機分為模型組(ICH組)、PDTC低濃度組(Plow組)和PDTC高濃度組(Phigh組),每組6只。Plow組和Phigh組分別在術(shù)后2 h腹腔注射100 mg/kg和200 mg/kg的PDTC,ICH組在同時點腹腔注射等體積的生理鹽水。
2.2神經(jīng)功能評分 術(shù)后24 h應(yīng)用改良Longa分級法[9]對各組大鼠進行神經(jīng)功能評分。評分標準如下:無神經(jīng)功能缺陷癥狀記0分;提尾時手術(shù)對側(cè)前肢不能伸直記1分;提尾時手術(shù)對側(cè)前肢屈曲記2分;行走時向手術(shù)對側(cè)轉(zhuǎn)圈,出現(xiàn)追尾癥記3分;行走時身體向手術(shù)對側(cè)跌倒記4分;不能自發(fā)行走或出現(xiàn)意識障礙記為5分。同時觀察并記錄大鼠的一般生存狀態(tài)。
2.3TUNEL法檢測神經(jīng)細胞凋亡 實驗結(jié)束時,將各組大鼠麻醉后,斷頭取腦組織,置于4%多聚甲醛中固定,常規(guī)乙醇梯度脫水,透明化后石蠟包埋,制備腦組織石蠟切片,切片厚度為5 mm。將腦組織石蠟切片常規(guī)脫蠟及水化處理后,加蛋白酶K消化30 min。PBS洗滌后浸入3%過氧化氫中封閉10 min;PBS洗滌后,吸去PBS,加TUNEL反應(yīng)液,于37 ℃暗濕盒中孵育60 min;PBS洗滌3次,吸去PBS,滴加Streptavidin-HRP(ICH工作液)于37 ℃暗濕盒中孵育30 min;PBS洗滌3次,滴加DAB顯色液,室溫顯色10 min,蘇木精復染。PBS洗滌后于光學顯微鏡下分析細胞凋亡情況。
2.4MDA含量及SOD活性的檢測 各組樣本經(jīng)相應(yīng)處理后,依據(jù)南京建成生物工程研究所提供的試劑盒檢測說明書檢測MDA含量及SOD的活性。
2.5Western blot檢測蛋白表達水平 取大鼠腦組織加適量裂解液,于冰上用勻漿器勻漿后離心取上清,得到各組蛋白樣品。所提的蛋白樣品采用BCA定量試劑盒進行定量分析后即可電泳。調(diào)整各組蛋白上樣總量至80 μg,按1∶4的體積比加入蛋白上樣緩沖液,98 ℃水浴變性5 min。上樣并行8%SDS-PAGE,待溴酚藍泳出分離膠后將蛋白電轉(zhuǎn)至PVDF膜(約90 min),加5%脫脂牛奶室溫封閉120 min。按說明書要求加入相應(yīng)的 I 抗,4 ℃孵育過夜,復溫后加TBST洗滌3次,每次5 min;再加入抗HRP標記的 II 抗,室溫孵育90 min,加TBST洗滌3次,每次10 min。于暗室中將HRP-ECL發(fā)光液灑在PVDF膜的蛋白面上激發(fā)熒光,經(jīng)壓片、顯影及定影后對所得的蛋白條帶行灰度分析。
所有實驗數(shù)據(jù)均采用SPSS 13.0軟件進行統(tǒng)計學分析。實驗結(jié)果以均數(shù)±標準差(mean±SD)表示,計量資料行t檢驗,計數(shù)資料行卡方檢驗或校正卡方檢驗,多組數(shù)據(jù)采用單因素方差分析(one-way ANOVA),并用Bonferroni法進行均數(shù)組間的兩兩比較,以P<0.05表示差異有統(tǒng)計學意義。
ICH術(shù)后可明顯見到各手術(shù)組大鼠精神萎靡、反應(yīng)遲鈍、活動減少,手術(shù)對側(cè)肢體及行為異常。手術(shù)組21只大鼠,造模成功19只;實驗中1只因術(shù)后感染死亡,1只不明原因死亡。假手術(shù)組大鼠實驗過程中整體狀態(tài)良好。
神經(jīng)功能評分結(jié)果顯示,ICH組大鼠的神經(jīng)功能評分明顯高于假手術(shù)組(P<0.05),說明ICH組大鼠出現(xiàn)明顯的神經(jīng)功能損傷;相較于ICH組,給予PDTC干預后,大鼠神經(jīng)功能評分有所降低(P<0.05),但Plow組和Phigh組2組之間無顯著統(tǒng)計學差異,見圖1。
Figure 1. The effect of NF-κB inhibitor PDTC on neurological function score of ICH rats. Mean±SD.n=6.*P<0.05vssham group;#P<0.05vsICH group.
圖1NF-κB抑制劑PDTC對ICH大鼠神經(jīng)功能評分的影響
TUNEL法結(jié)果顯示,假手術(shù)組僅可見個別細胞凋亡;ICH模型組可見大量神經(jīng)元中有點片狀或團塊狀的深棕色顆粒;應(yīng)用PDTC后,神經(jīng)細胞凋亡數(shù)目顯著降低,且Phigh組細胞凋亡數(shù)目顯著低于Plow組(P<0.05),見圖2。
MDA含量的檢測結(jié)果示,ICH模型組的MDA含量顯著高于假手術(shù)組;應(yīng)用PDTC后,腦組織中MDA的含量顯著降低,且Phigh組腦組織中MDA的含量顯著低于Plow組(P<0.05)。SOD活性的檢測結(jié)果示,ICH模型組中SOD的活性顯著低于假手術(shù)組;應(yīng)用PDTC后,腦組織中SOD的活性顯著升高(P<0.05),但Phigh組和Plow組2組間SOD的活性無顯著差異,見圖3。
Western blot 檢測的結(jié)果顯示,假手術(shù)組大鼠腦組織中有P65及caspase-3的基礎(chǔ)表達;ICH模型組大鼠腦組織中p-P65及cleaved caspase-3的蛋白水平顯著增加(P<0.05);與模型組相比,采用PDTC干預后,p-P65的蛋白水平顯著減少,可檢測到cleaved caspased-3的蛋白水平也顯著低于ICH模型組,且Phigh組中cleaved caspase-3的蛋白水平顯著低于Plow組(P<0.05),見圖4。
目前,研究腦出血的動物模型包括微自體血注入法、球囊充脹法、膠原酶注入法及自發(fā)性腦出血模型,本實驗中采用SD大鼠建立自體血注入ICH模型,經(jīng)動物行為學評價和神經(jīng)功能評分確認模型構(gòu)建成功,并用于后續(xù)實驗評價NF-κB抑制劑PDTC對ICH腦損傷及神經(jīng)細胞凋亡的作用。PDTC是NF-κB的特異性抑制劑,可抑制P65亞單位或IκB的降解阻止NF-κB的激活及轉(zhuǎn)位,并被廣泛用于心臟、腦及肺等相關(guān)疾病的研究中[10]。
Figure 2. The effect of NF-κB inhibitor PDTC on neural cell apoptosis of ICH rats detected by TUNEL assay (×400). Mean±SD.n=6.*P<0.05vssham group;#P<0.05vsICH group;△P<0.05vsPlowgroup.
圖2TUNEL法測定NF-κB抑制劑PDTC對ICH大鼠神經(jīng)細胞凋亡的影響
Figure 3. The effect of NF-κB inhibitor PDTC on oxidative stress in brain tissues of ICH rats. Mean±SD.n=6.*P<0.05vssham group;#P<0.05vsICH group;△P<0.05vsPlowgroup.
圖3NF-κF抑制劑PDTC對ICH大鼠腦組織氧化應(yīng)激水平的影響
Figure 4. The effect of NF-κB inhibitor PDTC on protein levels of p-P65 and cleaved caspase-3 in brain tissues of ICH rats. Mean±SD.n=6.*P<0.05vssham group;#P<0.05vsICH group;△P<0.05vsPlowgroup.
圖4NF-κB抑制劑PDTC對ICH大鼠腦組織p-P65和cleavedcaspase-3蛋白水平的影響
實驗結(jié)果顯示,與ICH模型組相比,給予低濃度及高濃度PDTC干預之后,兩組動物的神經(jīng)行為癥狀得到明顯改善,且神經(jīng)功能評分顯著降低。結(jié)果表明NF-κB抑制劑可改善ICH大鼠腦出血所致的神經(jīng)損傷,具有一定的治療價值。細胞的過度凋亡是ICH繼發(fā)性腦損傷的一個重要環(huán)節(jié),接著我們通過TUNEL法評價了ICH大鼠神經(jīng)細胞的凋亡情況及NF-κB抑制劑的作用。結(jié)果顯示,相比于假手術(shù)組,ICH模型組大鼠腦組織出現(xiàn)明顯的細胞凋亡,這與早期的實驗結(jié)果具有一致性;與ICH模型組相比,給予PDTC干預可減少神經(jīng)細胞的凋亡率,且高濃度的PDTC作用效果優(yōu)于低濃度的PDTC;結(jié)果表明NF-κB抑制劑可減輕ICH所致神經(jīng)細胞凋亡。
ICH所致的原發(fā)性和繼發(fā)性損傷會進一步導致血腫灶周血流量的減少。眾所周知,腦組織代謝旺盛,耗氧量極高,局部缺血易致自由基的大量生成及炎癥反應(yīng)的發(fā)生,這可能為細胞凋亡的誘因[11]。研究發(fā)現(xiàn),NF-κB在中樞神經(jīng)中的活性與氧化應(yīng)激狀態(tài)密切相關(guān)[12]。且有研究證實NF-κB抑制劑PDTC可以清除活性氧[13],故而本實驗進一步檢測了腦組織中MDA的含量及SOD的活性,以評價PDTC在調(diào)控ICH大鼠腦損傷及神經(jīng)細胞凋亡的過程中是否涉及到氧化應(yīng)激狀態(tài)的改變。結(jié)果顯示,與假手術(shù)組相比,ICH模型組大鼠腦組織MDA含量升高,SOD活性降低,氧化應(yīng)激水平升高;應(yīng)用NF-κB抑制劑PDTC后,2組動物腦組織中MDA含量顯著降低,而SOD活性升高。這一結(jié)果表明PDTC可改善ICH大鼠腦組織的氧化應(yīng)激狀態(tài),而這一作用可能也是減輕細胞凋亡的機制之一。細胞的凋亡可分為caspase依賴性和caspase非依賴性2種。本實驗進一步分析了caspase依賴性凋亡路徑中重要的效應(yīng)蛋白caspase-3表達的變化,結(jié)果顯示與假手術(shù)組相比,ICH模型組大鼠腦組織中cleaved caspase-3的蛋白水平顯著增加;應(yīng)用PDTC后, cleaved caspase-3的蛋白水平顯著降低,且高濃度組作用更明顯。結(jié)果提示PDTC調(diào)控ICH大鼠神經(jīng)細胞凋亡的機制涉及caspase依賴性凋亡信號通路,至于其上、下游涉及的其它信號分子及通路還須進一步的實驗驗證。
綜上所述,NF-κB抑制劑PDTC能減輕腦出血后繼發(fā)性腦損傷,且大劑量作用效果較好;其作用機制可能與降低MDA含量、提高SOD的活性進而抑制神經(jīng)細胞凋亡有關(guān)。
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Effects of NF-κB inhibitor on nerve injury in rats after intracerebral hemorrhage
FENG Lu, XUE Yue-hua, XU Zheng-bao, YAN Xiu-you, FANG Li-ren
(DepartmentofNeurosurgery,TaizhouCentralHospital,Taizhou318000,China.E-mail: 2039269058@qq.com)
AIM: To investigate the regulatory effect of nuclear factor-κB (NF-κB) inhibitor, pyrrolidine dithiocarbamate (PDTC), on nerve function and neural cell apoptosis in rats after intracerebral hemorrhage (ICH).METHODSSPF Sprague-Dawley rats were randomly divided into 4 groups with 6 rats in each group: sham group, ICH group, PDTC at low concentration (Plow) group and PDTC at high concentration (Phigh) group. Autologous blood injection was used to establish ICH model. After 2 h of surgery, the rats in Plowgroup and Phighgroup were intraperitoneal injected with PDTC at 100 mg/kg and 200 mg/kg, respectively, while rats in sham group and ICH group were injected with the same volume of saline. The neurological function score was classified with modified Longa grading method. TUNEL assay was used to detected the neural cell apoptosis, and the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) were measured. Furthermore, the protein levels of p-P65 and cleaved caspase-3 in brain tissues were determined by Western blot.RESULTSCompared with sham group, the rats in ICH group had higher neurological function score (P<0.05). After treatment with PDTC, the neurological function score was decreased (P<0.05), but no significant difference between Plowgroup and Phighgroup was observed. Compared with sham group, the number of apoptotic cells in ICH group was increased (P<0.05). After treatment with PDTC, the neural cell apoptosis was restrained, and the number of apoptotic cells in Phighgroup was lower than that in Plowgroup (P<0.05). Compared with sham group, the content of MDA was increased and the activity of SOD was decreased in ICH group (P<0.05). After treatment with PDTC, the content of MDA was decreased while the activity of SOD was increased, and the variation trend was more obvious in Phighgroup (P<0.05). Compared with sham group, the protein levels of p-P65 and cleaved caspase-3 in ICH group were increased (P<0.05). After treatment with PDTC, the protein levels of p-P65 and cleaved caspase-3 were decreased, and those in Phighgroup were lower than those in Plowgroup.CONCLUSIONNF-κB inhibitor PDTC plays a role in the se-condary brain injury after ICH, and the protective effect increases at the higher dose. The mechanism may be related to reducing MDA content and increasing SOD activity, and further inhibiting neural cell apoptosis.
Nuclear factor-κB inhibitor; Apoptosis; Intracerebral hemorrhage; Oxidative stress
1000- 4718(2017)12- 2278- 05
2017- 06- 09
2017- 09- 06
△通訊作者 Tel: 0576-81899120; E-mail: 2039269058@qq.com
R743.34; R363.2
A
10.3969/j.issn.1000- 4718.2017.12.027
(責任編輯: 林白霜, 羅 森)